PROJECT SUMMARY Manganese (Mn) is a trace nutrient that is essential for viability of organisms from bacteria to humans. Mn serves as an enzyme cofactor to help catalyze diverse chemical reactions. Mn also detoxifies and protects cells from reactive oxygen species. For these reasons, many pathogenic and symbiotic bacteria require Mn to survive in eukaryotic host tissues. However, excess Mn can be toxic. Therefore cells must carefully regulate the intracellular levels of Mn through homeostasis systems. In addition to a Mn importer and exporter, a small protein of only 42 amino acids called MntS helps control intracellular Mn levels in Escherichia coli. Despite its phenotypic connection to Mn homeostasis, the cellular role and mechanism of action of MntS are not understood. In part, the lack of understanding about MntS activity results from its small size. Small proteins (< 50 amino acids) are an emerging class of proteins that have unique structural and functional properties due to their short length. From the few existing studies, small proteins are thought to act by interacting with and regulating larger proteins, but little is known about their structure, binding activities, or how they evolve. Since it is one of the few small proteins for which a physiological role is known, MntS could serve as an example to provide insight into how these small protein genes arise in genomes and how they bind and control other proteins. Our overall goals are to uncover the mechanism of action of the model small protein MntS in Mn homeostasis and to use lessons learned from MntS to understand other small proteins. In Aim 1, we will test the hypothesis that MntS functions by inhibiting the MntP Mn exporter using genetic, biochemical, and gene expression experiments. We will carry out affinity co-purifications to detect interactions between MntS and MntP or other proteins. We will also use Western blotting to assess MntP stability in cells lacking mntS. In Aim 2, we will use newly-identified homology with a Mn importer to identify the essential amino acids for MntS function and characterize its subcellular localization. This will also provide insight into the origin of MntS as a model for small protein evolution. In Aim 3, we demonstrate that MntS forms one or more protein complexes in a Mn-dependent manner. We will characterize the putative MntS-MntS and MntS-Mn interactions using biophysical methods in vitro and the two-hybrid assay in vivo. These studies will give mechanistic information about Mn homeostasis in E. coli and related enterobacteria that may enable scientists to manipulate bacterial populations in eukaryotic hosts or the environment. In the long-term, these findings may help decrease disease by allowing us to eliminate pathogenic bacteria and aid growth of beneficial bacteria. Additionally, fundamental knowledge gained about small protein biochemistry and physiology from these studies will further understanding of how cells function and could be used to design novel small proteins with designer functions.